Natural Products Diversity of Marine Ascidians (Tunicates; Ascidiacea) and Successful Drugs in Clinical Development

The study of marine natural products (MNPs) is becoming ever more sophisticated and an increasingly collaborative effort between marine biologist, chemist and pharmacologist, which involves the discovery of new natural products to enter preclinical studies and clinical tests. Since the 1990s, several MNPs and their applications towards marine biotechnological and therapeutical potential were reported. Large numbers of bioactive compounds were dragged up from marine invertebrates, especially sponges, ascidians, bryozoans and molluscs in which some of them are approved by FDA and currently utilized in clinical trials [ 1 ]. Ascidians or sea squirts (Phylum: Chordata, Class: Ascidiacea) are also known as tunicates due to their external covering, found tied to rocks and high-current fields. There are approximately 3000 living species of ascidians were reported [ 2 ]. The production of chemical compounds is principally important for soft bodied ascidian species, which use secondary metabolites to deter predatory fishes, to compete for space, to control settlement and growth of microbial fauna and other fouling organisms. Ascidians represent the most highly evolved group of marine organisms commonly investigated for identification of MNPs and provide rich sources of bioactive secondary metabolite with promising potential biomedical applications [ 3 – 5 ]. So far a few novel compounds have been purified and characterized with a view of developing marine drug discovery. However, the most well known didemnins has been isolated from whole body homogenates of Caribbean ascidians belonging to the genus of Trididenium sp. [ 6 ]. More than 80% of new ascidians compounds contain nitrogen, and nearly 70% of nitrogenous metabolites are alkaloids [ 7 – 9 ]. These compounds often exhibit a range of biological activities such as cytotoxicity, antibiotic, immunosuppressive activities, inhibition of topoisomerases and cyclin kinases [ 10 ]. On the other hand, non-nitrogenous metabolites are fewer available in ascidians and also less significant. Hence, identification of the biogenetic origin of ascidian natural products is often challenging [ 11 ]. The first bioactive metabolite geranyl hydroquinone was isolated from the ascidian Aplidium sp. [ 12 ]; only 230 metabolites were isolated from ascidians during 1974–1992 [ 3 ]. At that time, a wide-ranging attention has focused on ascidians because of their biologically active metabolites and the chemical diversity of ascidians has become one of the most significant sources of MNPs. It has been demonstrated that marine ecosystems are essential producers of unusual chemical compounds and potent bioactivities [ 4 , 5 , 9 , 13 ]. Nonetheless, significant research in the area of marine pharmacology is a very recent origin, and also few products (or their analogues) have already reached the market as therapeutic drugs. Indeed, ascidian-derived natural products have yielded promising drug leads, among which ecteinascidin 743 (Yondelis ® ) and dehydrodidemnin B (Aplidin ® ) are in clinical usage for the treatment of specific cancers [ 14 , 15 ]. The research attempt on MNPs has not targeted all marine invertebrates equally. Ascidians are one of the most intensely studied organisms during the 21st century so that 572 secondary metabolites were reported from 1994 to 2014. This present study represented MNPs studied from family didemnidae (32%), polyclinidae (22%), styelidae and polycitoridae (11–12%) exhibiting the highest number of promising MNPs (Fig. 1 ). The distribution of chemistry class of ascidian MNPs are given in (Fig. 2 ). Close to 580 compound structures are here discussed in terms of their occurrence, structural type and reported biological activity. Anti-cancer drugs are the main area of interest in the screening of MNPs from ascidians (64%), followed by anti-malarial (6%) and remaining others (Fig. 3 ). The National Cancer Institute-United states estimate that approximately 1% of MNPs showing anti-tumor cytotoxicity properties as against only 0.01% amongst their terrestrial counterparts. Accordingly, finding MNPs research must being continued to progress to improve existing therapies and to develop novel cures. Fig. 1 Marine nature products studied from the family ascidian on 1994–2014 Fig. 2 Distribution of chemistry class of MNPs with high biomedical potential application studied from 1994 to 2014 Fig. 3 Distribution of drug classes of MNPs with high biomedical potential application studied from 1994 to 2014 This review focuses on the chemical diversity of marine ascidians. The recent research on MNPs has been surveyed at relatively frequent intervals [ 4 , 5 , 9 , 13 ]. Davidson [ 3 ] was published the first review on secondary metabolites derived ascidians from 1988 to 1993. Additionally, in contrast to the review of ascidian metabolites, the present study provides complete list of the compounds with biological activities; the primary focus of this article is addressed to s

Anti-microbial Activity Anti-bacterial Activity Four new sulphated alkanes/alkenes 2,6-dimethylheptyl sulphate, (4 Z ,7 Z )-4,7-decadienyl sulfate, (4 Z ,7 E )-4,7decadienyl sulphate, and (3 Z ,6 Z )-3,6,9-decatrienyl sulphate ( 50 – 53 ) were isolated from the hepatopancreas of the ascidian Halocynthia roretzi [ 44 ]. Compounds ( 50 – 53 ) showed 12 mm zones of growth inhibition against bacterial strain Vibrio alginoliticus and besides exhibited activity with fungal strains Mortierella ramaniana 10 mm zones at 0.2 mg/disk, respectively. Based on the results, Tsukamoto reported these simple sulfates may possibly play a physiological role in certain ascidian species. Two new anti-microbial metabolites, halocyntin and papillosin, were isolated from hemocytes of the solitary tunicate, H. papillosa were collected in Catalan coast, in Mediterranea Sea [ 45 ]. Both peptides showed potential zone growth inhibition against M. luteus (0.13–0.25 µg/mL) and E. coli (0.25–0.50 µg/mL), from this compound papillosin showed the potential value of anti-microbial activity compare to halocyntin. Amongst Ascidians, the family of Didemnidae have been described as a vital source of diverse MNPs with potent pharmacological properties. The potential important active metabolites such as didemnins, aplidine, and the tamandarins were reported from the family of Dideminidae [ 46 ]. Four new β -carboline based metabolites; Didemnolines (A–D) 54 – 57 were isolated from the ascidian Didemnum sp, collected in Northern Mariana Islands, United States [ 47 ]. Didemnolines C ( 56 ) shows 7 mm zone growth inhibition against E. coli and with Staphyloccocus aureus (9 mm). In addition, compound ( 54) shows potent cytotoxicity against human epidermoid carcinoma (KB cells) 0.28 µg/mL. Three new pyridoacridine alkaloids, isodiplamine, cystodytin K and lissoclinidine ( 58 – 60 ), and known alkaloids diplamine ( 61 ) and cystodytin J ( 62 ) were isolated from the ascidian Lissoclinum notti collected at Leigh Harbour, New Zealand [ 48 , 49 ]. Both compounds ( 58 – 59 ) were active against two marine bacteria Psychrobactor immobilis (1.5 µg/mL) and Planococcus citreus (1.5 µg/mL). Compounds ( 60, 61 ) showed zones of growth inhibition against B. subtilis (8, 9 mm) and E. coli (6, 3 mm) at the highest concentration 120 mg of pyridoacridine alkaloid compound, on to 6 mm paper disc. Furthermore, Pyridoacridine alkaloids inhibited the growth of fungal T. mentagrophytes (9, 6 mm), lissoclinidine was completely inactive against C. albicans and diplamine showed potent inhibition (12 mm), respectively. Eudistomins W and X ( 63 – 64 ) were isolated from the colonial ascidian Eudistoma sp., collected in on mangrove roots at Chuuk, Micronesia [ 50 ]. Eudistomins W and X showed a moderate growth inhibition against bacterial strains B. subtilis (17, 18 mm), E. coli (15, 20 mm) and S. aureus (11, 12 mm) at concentration 5 and 10 µg per disc, respectively. In addition, Eudistomins W, X inhibited the zone growth against fungi C. albicans (13, 18 mm) at similar loading doses. Marine alkaloids, Lissoclibadins ( 65 – 67 ), lissoclinotoxins E and F ( 68, 69 ) were isolated and reported from the ascidian Lissoclinum cf . badium , collected at coral reef station Manado, Indonesia [ 51 , 52 ]. Both compounds ( 65, 66 ) inhibited the growth of the marine bacterium Ruegeria atlantica (15.2 mm and 12.2 mm at 20 mg/disc), and compound ( 66 ) exhibited antifungal activity towards Mucor hiemalis (13.8 mm at 50 mg/disk). In addition, Lissoclibadins 1–3 have also showed cytotoxic against HL-60 (IC 50 = 0.37, 0.21, and 5.5 µM). Furthermore, four new polysulfur aromatic alkaloids, Lissoclibadins ( 70 – 73 ) were isolated from the same ascidian [ 53 ]. Compounds ( 70 – 73 ) inhibited the colony formation of Chinese hamster V79 cells with EC 50 values of 0.71, 0.06, 0.06, and 0.17 µM, respectively. Moreover, compounds ( 70 – 73 ) exhibited poor anti-microbial activity against E. coli , Staphylococcus aureus , and Saccharomyces cere v isiae . Simon-Levert et al. [ 54 ] isolated five marine meroterpenes which include two new meroterpenes, methoxyconidiol and didhydroconicol ( 74, 75 ) and three known derivatives, cordiachromene A ( 76 ), epiconicol ( 77 ) and conidone ( 78 ) from the Aplidium aff. Densum, collected in Masirah Island, Oman. These meroterpenes ( 74, 75 ) have inhibited the zone growth (MIC) against bacterial strains with E. coli (>2 µM) and M. luteus (>2 µM, 0.51 µM). Moreover, Compound ( 74 ) was a potential source of MNPs affecting the reproduction processes of sea urchin Sphaerechinus granularis and Paracentrotus lividus , inhibited the cleavage fertilised eggs. It interrupts M-phase development and completely blocks cytokinesis without any effect on DNA replication, most likely affecting microtubule dynamics [ 55 ]. Five new serinolipid derivatives, Shishididemniols A–E ( 79 – 83 ) were isolated from Didemnum sp. collected in Japan [ 56 ]. Compounds ( 80 – 83 ) ex

Anti-fungal active compound (2 S ,3 R )-2-Aminododecan-3-ol ( 131 ) was isolated from the ascidian Clavelina oblonga collected in Brazil [ 77 ]. Compound (131) showed remarkable MICs against Candida albicans (0.7 µg/mL) and C. glabrata (30 µg/mL). Moreover, 2-amino-alkanols and their unsaturated derivatives have been repeatedly reported in marine ascidians and sponges [ 78 ]. Furthermore, new metabolites of cadiolides A, B ( 132 – 133 ) were isolated from the ascidian Botryllus sp. [ 79 ]. Additionally, Won et al. [ 68 ] reported cadiolides E–H ( 102 – 105 ) from another ascidian Synoicum sp. Both compounds ( 102, 105 ) showed a potent anti-fungal activity zone growth inhibitory with C. albicans (IC 50 values of 7.62, 10.36 µM) [ 80 ]. Searle and Molinski [ 81 ] isolated the anti-fungal 2-amino alcohol ( 134 ) and their derivatives ( 134a, b ) from the Australian Didemnum sp. Compound ( 134) was related to sphingosine ( 135 ), a widely distributed amphiphilic amino alcohol. A synthetic peptide halocidin was isolated from Halocynthia aurantium and showed remarkable antifungal activity against C. albicans (1–4 µg/mL). Hence, halocidin peptide was considered as a potential source for the development of new antibiotic resistant mechanisms [ 82 ].

An unusual sulfated mannose, homopolysaccharide ( 143 ) also known as kakelokelose, was reported from the mucous secretion of the ascidian D. molle collected from Pohnpei, Micronesia and Manoda, Indonesia [ 85 ]. Compound ( 143 ) showed a remarkable anti-HIV activity determined 100% potential to inhibit infection with CEM cells by HIV strain RF at 0.3 µg/mL, whereas no cytotoxicity against CEM cells at concentrations 15 µg/mL. Furthermore, five lamellarins, the 20-sulfates of lamellarins B, C, L, lamellarin G 8-sulfate and lamellarin Z ( 144 – 148 ) were isolated from Didemnum chartaceum from the Great Barrier Reef, Australia. Unusually long relaxation times were observed for certain signals in their 1 H NMR spectra [ 86 ]. During further investigation, Reddy et al. [ 87 ] reported lamellarin α 20-sulfate ( 149 ) from an unidentified ascidian, collected in Indian waters. Compound ( 149 ) showed moderate inhibition of anti-HIV activity against HIV-1 protease (IC 50 16 µM). An anti-retro viral metabolite, cyclodidemniserinol trisulfate ( 150 ) was reported from the ascidian Didemnum guttatum collected at Ngerchaol Island, Palau. Compound ( 150 ) is very similar to didemniserinolipid A ( 151 ), reported from an Indonesian Didemnum sp. [ 88 ], though few significant variations between these two chemical structures, like the presence of an additional ring containing a glycine unit and the presence of sulfate groups, were observed. Moreover, Gonzalez et al. [ 88 ] also reported didemniserinolipids B and C ( 152 – 153 ) from the same ascidian species. Anti–HIV activity of compound ( 150 ) showed a modest inhibition against HIV-1 protease (IC 50 60 µg/mL) and with MCV topoisomerase IC 50 72 µg/mL. Compound ( 150 ) showed no selectivity for integrase inhibition. During the screening of anti-HIV agents from marine organisms, Donia et al. [ 89 ] reported two new cyclic hexapeptide alkaloid including mollamides B ( 154 ) and C ( 155 ), with known peptide keenamide A ( 156 ) from the tunicate Didemnum molle collected in Manado Bay, Indonesia. Compound ( 154 ) showed in vitro anti–HIV activity against HIV-1 human PBM cells (EC 50 48.7 µM). Lu et al. [ 90 ] isolated two new Thiazoline peptides, mollamides E and F ( 157 – 158 ) and one new a Tris-Phenethyl urea, molleurea A ( 159 ) from the same ascidian D. molle collected in Papua New Guinea. Compounds ( 157 – 159 ) were tested for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. Mollamide F ( 158 ) showed a moderate activity against both bio-assays with IC 50 values of 39 and 78 µM, and molleurea A ( 159 ) was effective in the cytoprotective cell-based assay (IC 50 60 µM) (Structure 4 ). Structure 4 Anti-fungal potential compounds ( 131 – 159 )

New pyridoacridine alkaloids, 12-deoxyascididemin ( 189 ), and two well-known analogues, ascididemin ( 5 ) and eilatin ( 190 ) were isolated from the ascidian Polysyncraton echinatum collected in North West of Farquharson Reef, Australian coast [ 109 ]. Compounds ( 189, 5, 190 ) had shown potent anti-trypanosomal activity against Trypanosoma brucei brucei (IC 50 0.077, 0.032, 1.33 µM). Additionally, compounds ( 189, 5) showed a moderate cytotoxicity against HEK293 cell line (IC 50 7.63, 1.48 µM). Simarro and co-workers had reported 70 million populations of 37 sub-saharan African countries were affected deadly infectious disease [ 110 ], hence it is urgently required to develop new effective drugs to fight against African sleeping sickness (Structure 6 ). Structure 6 Anti-trypanosomal potential compounds ( 5 , 189 , 190 )

Two new tricyclic thiazine-containing quinolinequinone alkaloids, ascidiathiazones A and B ( 177 , 206 ) were reported from Aplidium sp. collected in New Zealand ascidian coast. Compounds ( 177 , 206 ) showed a potential inhibition against human neutrophils (IC 50 1.55, 0.44 µM), respectively [ 117 ]. Furthermore, rubrolide O ( 207 ) was reported from the Synoicum sp. collected in New Zealand coast. Rubrolide O is a new member of rubrolide family exist a mixture of E/Z -isomers. Compound ( 207 ) showed a modest inhibition against human neutrophil free radical release (IC 50 35 µM) [ 118 ]. Belmiro and co-workers reported anti-inflammatory properties of a dermatan sulfate similar to mammalian heparin from the ascidian Styela plicata , collected in Brazil coast [ 119 , 120 ]. The molecular characterization of this analogue have been tested by in vivo rat colitis model, showing a significantly decreased lymphocyte and macrophage recruitment as well as TNF- α , TGF- β , and VEGF production in the inflamed rat colon at concentration 8 mg/kg per day. New anti-inflammatory meroterpenoids, 2-geranyl-6-methoxy-1,4-hydroquinone-4-sulfate ( 161 ) and scabellone B (163) showed a moderate potent anti-inflammatory activity (21, 125 µM), and selective ability to inhibit neutrophil respiratory burst proves that meroterpenoid sulfates may have potential for developing novel marine drugs for treatment of inflammation [ 96 ]. Biologically active amino acid derivatives, herdmanines A–D ( 208 – 211 ) were reported from the ascidian Herdmania momus , the unusual D-form of arginine present in herdmanines A–C [ 121 ]. Both compounds ( 210, 211 ) showed adequate suppressive effects on the production of NO (IC 50 96 and 9 µM) and these compounds have the potential to inhibit the mRNA expression of iNOS. Additionally, compound 211 exhibited a strong inhibition of mRNA expression of pro-inflammatory cytokines IL-6. Further investigations are needed to search for potential anti-inflammatory MNP from the same ascidian. A series of anti-diabetic amino acid derivatives new congeners herdmanines E–L ( 212 – 220 ) and (−)-( R )-leptoclinidamine B were reported [ 122 , 123 ]. Indoleglyoxylyl derivatives, herdmanine K showed strong PPAR- γ activation in rat liver cells (Ac2F) 1 and 10 µg/mL in a cell-based luciferase reporter assay. Anti-inflammatory activity of methanol crude compounds extracted from the ascidian Eudistoma virde was exhibited modest anti-inflammatory activity at various concentration up to 200 mg/kg compare to the positive control Diclofenac [ 124 ]. The biological activity is similar to that one of other ascidians Synoicum sp. and Pycnoclavella kottae [ 48 , 118 ] (Structure 8 ). Structure 8 Anti-inflammatory potential compounds ( 161 , 163 , 177 , 206 – 220 )

The drug discovery of anti-cancer activity has been the main area of interest in the field of natural product chemistry. Tunicates are particularly successful to yielding anti-tumor compounds; several MNPs were under various clinical trials in Europe and USA. With the continuous searching of effective and targeted anti-cancer drugs, in this section we explore the unique perspectives, novel biomolecules isolated from marine ascidians with promising pharmacological potential source of anti tumor/anti-cancer and anti-proliferative activity. Three new linear cytotoxic tripeptides, virenamides A–C ( 237 – 239 ) have reported from the Didemnid ascidian Diplosoma virens collected in Great Barrier Reef, Australia [ 134 ]. Virenamide A ( 237 ) exhibited effective cytotoxicity against various cultured cells with P388 (IC 50 2.5 µg/mL), and showed moderate cytotoxicity against A549, HT29, and CV1 cells line (IC 50 10 µg/mL). Compounds ( 238 – 239 ) showed modest cytotoxicity against P388, A549, HT29, and CV1 cells. Additionally, virenamide A ( 237 ) showed topoisomerase II inhibitory activity (IC 50 2.5 µg/mL). Lindsay and co-workers have been isolated pyrido [2, 3, 4- kl ] acridine-based alkaloid ascididemin 2 ( 55 ) from the Didemnum sp. [ 135 ]. Ascididemin 2 showed remarkable significant cytotoxicity against human tumor cells; P-388 mouse leukemia (IC 50 0.4 µM), human colon HCT II6 (IC 50 0.3 µM), and with human breast MCF7 (IC 50 0.3 µM), respectively. In continuing search of MNPs to find anti-tumor drugs, Koulman et al. [ 136 ] was tested the cytotoxicity of colonial ascidian Didemnum lahillei , Aplidium glabrum, Molgula manhattensis collected in Lake Grevelingen and Oosterschelde estuary, Netherland. The crude extract of A. glabrum and M. manhattensis showed modest cytotoxicity against colon adenocarcinoma cells (IC 50 5, 8 µg/mL). The crude compound of D. lahillei showed moderate cytotoxicity against colon adenocarcinoma cell lines (IC 50 33 µg/mL) and with small cell lung carcinoma (IC 50 49 µg/mL), respectively. Two new dimeric alkaloids, lissoclinotoxins E, F ( 68, 69 ) were isolated from the Philippine didemnid ascidian collected in Sabtang Reef, Batanes Islands [ 137 ]. Both compounds ( 68, 69 ) showed potent cytotoxicity growth inhibition against MDA-MB-468 human breast carcinoma cell line (IC 50 2.3, 1.5 µg/mL), respectively. Lissoclinotoxins E and F shown potent cytotoxicity towards the MDA-MB-468 (PTEN −/− ) and the MDA-MB-435S (PTEN +/+ ) cell lines. Lissoclinotoxin F showed a threefold higher cytotoxicity against the PTEN deficient cell line. During the chemical investigation of Brazilian tunicates Didemnum granulatum, new alkaloid didemnimide E ( 242 ), and a new G2 checkpoint inhibitor and known marine alkaloids didemnimides A ( 240 ) and D ( 241) were reported [ 138 ]. Further investigation, authors worked in the same ascidian species was reported isogranulatimide ( 37 ) with two minor compounds granulatimide ( 243 ) and 6-bromogranulatimide ( 244 ) [ 139 ]. Both compounds ( 243, 37 ), showed activity as an inhibitor of the G2 cell cycle checkpoint in vitro and combined with DNA damaging p53 cancer cells (IC 50 1–1.8 µM). Anti-cancer effective marine alkaloid, mollamides B, C ( 154, 155 ) reported from Didemnum molle. Mollamide B ( 154 ) showed modest cytotoxicity against non-small cell lung cancer H460 (IC 50 29 µM), CNS cancer cell line SF-268 (IC 50 42 µM), and breast cancer cell line MCF7 (IC 50 44 µM) at concentration 100 µM [ 89 ]. Mollamide C exhibited poor cytotoxicity with murine leukaemia L1210 cells and human breast MCF-7 cells lines. From the Australian ascidian A. confluata , marine alkaloid, aplidiopsamine A ( 178 ) was reported, compound ( 178 ) exhibited moderate cytotoxicity against HEK-293 cell line (IC 50 120 µM) at higher doses [ 102 ]. From the crude extracts of tunicate Leptoclinides dubius, the first Leptoclinides -derived indole compounds: N -(1 H -indolyl-3-carbonyl)- d -arginine ( 7 ), N -(6-bromo-1 H -indolyl-3-carbonyl)- l -arginine ( 8 ), N -(6-bromo-1 H -indolyl-3-carbonyl)- l -histidine ( 9 ) and N -(6-bromo-1 H -indolyl-3-carbonyl)- l -enduracididine ( 10 ) were reported by García et al. [ 17 ]. Remarkably, the major group of these compounds are nitrogen-containing compounds with aromatic heterocycles. Compounds ( 7 – 9) showed 80–100% inhibition of cytotoxic against P-388 cells and KB cells at 10 µg/mL. Furthermore, four new meroterpenoids such as conidione ( 245 ), conicol ( 246 ), 2-[(1′ E )-3′-methoxy-3′,7′-dimethylocta-1′,6′-dienyl]benzene-1,4-diol ( 247 ) and conitriol ( 248) , and five known compounds and their derivatives ( 76 , 249 – 253 ) were isolated from the Mediterranean ascidian Aplidium conicum collected in Tarifa Island, Spain [ 140 ]. In further investigation of the same ascidian species which was collected from Alghero, Italy; two new unique alkane derivatives, conicaquinones A (254) and B ( 255 ) were reported by Aiello et al. [ 140 ]. Compound

Marine ecosystem forms an important source of unique compounds with high structural uniqueness and incomparable chemical properties. At the core of MNPs discovery is the identification procedure and NMR stays on the most useful tool [ 310 ]. At this time, natural products chemistry research is progressing a dynamic comeback in the modern drug discovery. Relatively more advances have taken place concerning the inherent capabilities of NMR apparatuses, able to reduce experiment times and increase sensitivity toward more efficient analyses of novel compounds present in µM level [ 311 ]. The advance of high resolution magic angle spin NMR (HR-MAS NMR) probes is most useful to analyse intact tissues. Nevertheless, while (HR-MAS NMR) is incorporated in food chemistry where both primary and secondary metabolites are of importance, it has not been yet widely introduced in natural products chemistry [ 312 ]. Queiroz Junior et al. have impressively demonstrated the significance of the synergy between NMR hardware and innovative pulse sequences. It is the first report that an ultrafast COSY pulse sequence is applied to a hyphenated LC–NMR separation of crude extracts (Ex. three natural flavonoids; naringin, epicatechin and naringenin). The detection volume was only 60 mL, while two scans have proven sufficient to get spectra with optimized resolution and sensitivity. This application portrays the generality of ultrafast methodologies in natural product chemistry, placing LC–NMR as an effective analytical tool [ 313 , 314 ]. For example, NMR experiments such as DOSY and JRES were also very useful routine methodology for unraveling new chemical structures. NMR spectroscopy tirelessly continues leading this procedure. Furthermore, decisive chemical structural information could be derived from statistical interpretation methods applied in metabolomics such as statistical heterospectroscopy (SHY) [ 315 ], statistical total correlation spectroscopy (STOCSY) [ 316 ], heteronuclear single quantum correlation spectroscopy (HSQC), heteronuclear multible-bond correlation Spectroscopy (HMBC), Subset optimization by reference matching (STORM) [ 317 ], cluster analysis statistical spectroscopy (CLASSY) [ 318 ], multivariate statistical analysis of natural products fragments [ 319 ]. Nearly, dereplication analysis is necessary for computational support of data handling, processing and for structure elucidation purpose. Whereas user-friendly and sophisticated software packages are easily reached for effective data mining, they are not widely used for dereplication purposes in marine natural product chemistry [ 320 ]. However, the structure elucidation is most challenging task mainly due to uniqueness of natural products and unexpected spectral patterns and the residual complexity frequently noticed. For instance, prediction and simulation software such as PERCH, in combination with 1 H iterative full spin analysis (HiFSA approach), given an accurate distinction of natural products with nearly identical NMR spectra. As, proposed by the authors as a tool for puzzling qNMR analyses, it could be an alternative source of dereplication data [ 321 ]. Moreover, computer-assisted structural elucidation (CASE) is the techniques of using software that allows users to input their NMR data, and through matching algorithms to generate all possible molecular structures. For this purpose, software used are mainly the Structure Elucidator by ACD Labs, StrucEluc and CCASA [ 322 – 324 ]. Nevertheless, the success of these approaches is dependent on the quality of the spectra to be processed and the efficacy of the algorithms used. Furthermore, the software used present an inherent dependence on the databases from which data are extracted. Unfortunately, NMR databases dedicated to NPs appear as in-house, fragmented attempts, or are chemical group/organism/NMR experiment/solvent, among others, specific. For instance, MarinLit, and AntiBase, specialize in marine, fungal and microorganism NPs, NAPROC-13 is based on 13C resonances [ 325 ], while recently compiled TOCCATA uses 13C-labeled NPs [ 326 ]. Commercial NMR databases are limited to few vendors, like the SpecInfo database of Wiley and Bruker’s NMR database [ 327 , 328 ].

In a new marine drug discovery approach, structurally more complex MNPs was moved the next step from discovery to clinical trials based upon the strength of the industrial reproducibility. The discovery of novel marine drugs will continue to diversify. Research laboratories, academic entrepreneurs, and innovative biotechnology industries will play major role in the discovery of novel marine drugs. The industrial collaboration program between natural products researchers and biotechnology industries will be instrumental to the primary clinical trials and mechanism of action studies crucial to provide the compelling preclinical data to create ample interest from larger pharmaceutical companies to lead and support for drug discovery program of MNPs. Also, it is essential to identify molecular targets for strongly active biomolecules and the ability to synthetically produce novel biomolecules to progress and discover new drugs. A recent development in biotechnological approach is revolutionizing the field of natural products chemistry. It is worth to mention here, during the isolation process was able collect only tiny amount strongly active biomolecules. It is very hard to collect in required quantity, the advanced techniques and the availability of new methods in chemical and biological synthesis have provided access to even the most complex of drug lead structures. High advanced developments in analytical tools and molecular biological science facilitate to identify the primary producers of secondary metabolites from symbiotic assemblage, and enable researchers to further explore the marine microbial chemical diversity for drug like biomolecules. Furthermore, those advances aide the characterization of several biosynthetic gene clusters and pathways and ultimately allow for their manipulation. Marine microbial chemical diversities are now easily explored drug like compounds using effective biosynthetic genetic engineering and in vitro multi enzyme synthesis methods [ 342 ]. Remarkably, David Hopwood’s group [ 343 ] has biosynthesized anti-biotic compound actinorhodin from Streptomyces coelicolor by cloning and heterologous expression of an entire biosynthetic pathway. Using genetic engineering techniques, Donia et al. [ 344 ] were prenylated anti-tumor compound trunkamide which is previously isolated in ascidian and different genera of cyanobacteria in E. coli culture. Didmnid ascidian species specificity of symbiosis and secondary metabolism in ascidian species were reported [ 344 ], collected in Florida coast. In this study, species specific and location-specific components were observed in Dideminid ascidian microbiomes and metabolomes. It is concluded that the biotechnological approaches in the field of natural products chemistry is more useful for sustainable supply of high quality marine drugs.